Crystal structure of the flavin reductase of Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH) and identification of amino acid residues underlying its regulation by aromatic ligands.

Yuenyao A, Petchyam N, Kamonsutthipaijit N, Chaiyen P, Pakotiprapha D
Arch Biochem Biophys 653:24-38 (2018 Sep 1)

PMID: 29940152 doi: 10.1016/j.abb.2018.06.010

Submitted to SASBDB: 2018 Feb 28 Published in SASBDB: 2018 Jun 27

SASDDT3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component: 2 mg/ml of wild-type C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase, reductase component dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 6.9 nm VolumePorod 94 nm3

SASDDU3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component: 4 mg/ml of Wild-type C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase, reductase component dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.1 nm VolumePorod 95 nm3

SASDDV3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component: 8 mg/ml of Wild-type C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase, reductase component dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.2 nm VolumePorod 95 nm3

SASDDW3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component: 2 mg/ml of Wild-type C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase, reductase component dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.8 nm Dmax 8.7 nm VolumePorod 85 nm3

SASDDX3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component: 4 mg/ml of Wild-type C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase, reductase component dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.7 nm Dmax 8.6 nm VolumePorod 95 nm3

SASDDY3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component: 8 mg/ml of Wild-type C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase, reductase component dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.7 nm Dmax 8.9 nm VolumePorod 102 nm3

SASDDZ3 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of H170A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component H170A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.0 nm VolumePorod 97 nm3

SASDD24 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of H170A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component H170A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.1 nm VolumePorod 116 nm3

SASDD34 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of H170A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component H170A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.0 nm VolumePorod 98 nm3

SASDD44 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of H170A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component H170A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.6 nm Dmax 8.6 nm VolumePorod 85 nm3

SASDD54 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of H170A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component H170A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.8 nm Dmax 9.0 nm VolumePorod 107 nm3

SASDD64 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of H170A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component H170A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.7 nm Dmax 9.0 nm VolumePorod 106 nm3

SASDD74 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of S172A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 6.7 nm VolumePorod 98 nm3

SASDD84 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of S172A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.1 nm VolumePorod 111 nm3

SASDD94 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of S172A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.2 nm VolumePorod 106 nm3

SASDDA4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of S172A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.7 nm Dmax 8.2 nm VolumePorod 83 nm3

SASDDB4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of S172A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.8 nm Dmax 8.6 nm VolumePorod 98 nm3

SASDDC4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of S172A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.8 nm Dmax 8.6 nm VolumePorod 105 nm3

SASDDD4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of N174A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component N174A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.0 nm VolumePorod 93 nm3

SASDDE4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of N174A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component N174A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.6 nm Dmax 7.6 nm VolumePorod 96 nm3

SASDDF4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of N174A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component N174A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.2 nm VolumePorod 102 nm3

SASDDG4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of N174A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component N174A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.8 nm Dmax 8.7 nm VolumePorod 94 nm3

SASDDH4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of N174A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component N174A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.8 nm Dmax 8.8 nm VolumePorod 111 nm3

SASDDJ4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of Y207A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component Y207A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 6.9 nm VolumePorod 104 nm3

SASDDK4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of Y207A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component Y207A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.5 nm Dmax 7.1 nm VolumePorod 111 nm3

SASDDL4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of Y207A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component Y207A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.5 nm Dmax 7.3 nm VolumePorod 109 nm3

SASDDM4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of Y207A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component Y207A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.3 nm VolumePorod 104 nm3

SASDDN4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of Y207A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component Y207A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.5 nm Dmax 7.2 nm VolumePorod 122 nm3

SASDDP4 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of Y207A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component Y207A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 1 mM HPA, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2017 Mar 7 RgGuinier 2.4 nm Dmax 7.2 nm VolumePorod 106 nm3